JPK Tech Force Spectroscopy.14 2 PDF

A practical guide to AFM force spectroscopy and data analysis
A) C)
"Fishing" for single molecules
The atomic force microscope (AFM) is best known for its
high-resolution imaging capabilities, but it is also a B) Cantilever
powerful tool for sensitive force measurements. The AFM
can be used to "fish" for single molecules and investigate
their structure and biological function. The study of Tip
interaction forces with the AFM has led to deeper Fig 1. Scanning electron microscopy images of typical pyramidal
(A) and cone-shaped (B) AFM tips and an optical microscope
understanding of many biological and physical processes
image of an AFM cantilever and tip (C).
down to the single molecule level.
The applications of these experiments range from nano-
The AFM tip is able to probe an extremely small interaction mechanical investigations of elastic properties to protein
area (using a tip radius in the range of 5-50 nanometers), unfolding and investigations of single chemical bonds [1].
and this gives it a high sensitivity to small forces. To make This technical report introduces the data collected in force
these forces accessible, the tip is suspended on an very curves and the standard steps that are used in converting
soft spring, the AFM cantilever (images in Figure 1). These the data for a quantitative force analysis.
cantilevers are usually silicon or silicon nitride beams, 100-
200 microns long, and allow forces in the pico-Newton Overview
range to be measured. This corresponds to the order of The schematic diagram in Figure 2 shows the movement
magnitude of forces that are required to separate receptors of the cantilever and tip during the force spectroscopy
from ligands or to break a single hydrogen bond. experiment towards the sample (the approach part) and
then away again (the retract part). The image series
In AFM imaging modes, the cantilever is usually scanned reflects different points in the approach and retract cycle,
over the surface to produce a three dimensional image of the lateral position of the cantilever is usually constant. A
the surface. In AFM force spectroscopy experiments, the flexible software interface allows for waiting times at
cantilever and tip are moved directly towards the sample different points of the movement for example at the
until they are in contact with it, and then retracted again, sample surface to give time for molecules to adsorb to the
while the interaction between the tip and sample is cantilever tip or at the end of the retract movement, to give
measured. This may then be repeated at different the surface time to recover.
locations to build up a map of the tip-surface interaction, or
can be repeated at the same point to give a full statistical Table 1 gives a summary of interactions that have been
understanding of the interaction. measured using the atomic force microscope, to give an
idea of the experiments that are possible and the parts of
the force curves where particular interactions are seen.
Approach Retract
Fig. 2 Schematic diagram of the vertical tip movement during the approach and retract parts of a force spectroscopy experiment.
page 1/8
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Approach
Tip far away No interaction
(10 - 100 microns)
Tip approaching Electrostatic forces

(few microns) Long-range interactions from adsorbed molecules, e.g. polymer brush
Tip close to surface Van der Waals

Capillary forces (in air)
(nanometers to atomic DLVO/screened electrostatics (in aqueous solutions)
distances) Chemical potential
Magnetic
Solvation forces (water layering)
Contact
Tip indenting sample Stiffness (Young's modulus, elastic response)
Viscoelastic response (variable rates or indentation depth)
Measurement of active forces (e.g. generated by cells)
Cell
Retract
Tip lifting off surface Adhesion:
(few atomic distances to Non-specific (including chemical affinity, surface coatings)
nanometers) Ligand-receptor (e.g. antibody-antigen)
Receptor-
DNA hybridisation (e.g. matched or mismatched pairs)
ligand
Cell surface interactions
Tip further away Stretched molecules between tip and surface:

(nanometers to Protein unfolding, pulling out of membranes
hundreds of Entropic elasticity
nanometers) DNA stiffness, structural transitions and "melting"
Other conformational changes in stretched molecules, DNA or
e.g. chair-to-boat transition in sugar rings polymer
Other stretched attachments e.g. membrane tethers formed on cells
Tip far from surface Connections broken between the tip and surface, no further interaction.
(1-5 microns) Adhesion strength can be measured between attached molecules and the surface when the
attachments break.
Table 1. Overview of some of the interactions measured at different points during a force spectroscopy cycle.
page 2/8
The forces experienced by the cantilever as it approaches mica. Approach (red) and retract (blue) curves are both
from several microns above the surface can give plotted on the same axes.
information about long-range interactions, such as As the cantilever approaches the surface, initially the
electrostatic effects. As the cantilever comes close to the forces are too small to give a measurable deflection of the
surface (nanometers or atomic distances), shorter-range cantilever, and the cantilever remains in its undisturbed
forces, such as Van der Waals or capillary forces can be position. At some tip-sample distance, the attractive forces
measured. When the sum of these forces is attractive, the (usually Van der Waals, and capillary forces in air)
tip may jump into contact with the surface, an effect overcome the cantilever spring constant and the tip jumps
referred to as snap-in. into contact with the surface. Once the tip is in contact with
the sample, it remains on the surface as the separation
Once the cantilever has made contact with the surface, it between the base of the cantilever and the sample
may be pushed into the surface with some force, in which decreases further, causing a deflection of the cantilever
case the viscoelastic properties of the sample can be and an increase in the repulsive contact force.
investigated, to give the Young's modulus, or stiffness for
different strain rates or maximum applied forces. When Repulsive contact
(indentation)
the cantilever is pulled away from the surface, adhesion
forces can be measured, and single molecules attached
between the tip and surface can be stretched. Jump to contact Approach
Understanding simple force curves

The data from an experiment is often displayed as a simple
Retract
x-y plot. The height positions for the approach or retract of
the cantilever are usually chosen as the x-axis, and the
cantilever property that is being measured is the y-axis.
Adhesion
This is usually the deflection of the cantilever, which can
give a direct measure of the interaction force. Sometimes
other signals are used for example current or
Fig. 3 Plot of approach (red) and retract (blue) curves for a contact
fluorescence, and particularly in dynamic AFM
mode cantilever and clean mica in air.
spectroscopy modes the amplitude or phase of the
cantilever oscillation. These "force-distance" plots are often
As the cantilever is retracted from the surface, often the tip
referred to as force curves. Unfortunately, there are many
remains in contact with the surface due to some adhesion
different conventions for how the data is displayed, so data
and the cantilever is deflected downwards. For
from different sources may look quite different.
measurements in air, such as the one shown in Figure 3,
there is usually adhesion from capillary forces between the
The most direct way to plot the data shows the movement
tip and sample.
of the piezo during the force curve (as a distance) against
the deflection of the cantilever. The deflection is measured
Hydrophilic surfaces are covered with a thin (few
by an optical beam deflection setup which delivers an
nanometers) water layer in ambient conditions, and often
electrical signal (in Volts, as the signal from the
the tip and sample are both hydrophilic. In this case, the
photodiode) that is proportional to the cantilever deflection.
water layers can join when the tip and sample are close
The example shown in Figure 3 reflects a typical
together, forming a capillary neck between them. When the
interaction for an uncoated cantilever in air approaching a
cantilever is retracted from the sample this combined water
hard, incompressible hydrophilic surface such as glass or
layer causes a strong adhesion. At some tip-sample
distance, the force from the cantilever will be enough to
page 3/8
overcome the adhesion, and the tip will break free. Often repulsive contact region is marked on the figure, and the
the adhesion between the tip and the sample in water is calculation in this case gave a sensitivity of 22 nm/V. This
actually reduced, since there is no longer this special water value can then be used to convert the deflection data along
surface layer interaction, as can be seen from the reduced the whole curve into nanometers.
adhesion in the curves in Figure 4.
Calibration of the cantilever deflection Approach

The deflection of the cantilever spring is directly
proportional to the tip-sample interaction force, but there Change in Retract
deflection
are two measurements required to convert the (Volts)
photodetector signal into a quantitative value of force. The
first stage is to calibrate the distance that the cantilever
actually deflects for a certain measured change in Baseline for deflection values
Change in piezo
photodetector voltage. This value depends on type of height (nm)
cantilever, but also on the optical path of the AFM
detection laser, and will be slightly different each time the
cantilever is mounted in the instrument. Once the
deflection of the cantilever is known as a distance, x, the
Fig. 4 Plot of approach (red) and retract (blue) curves in water.
spring constant, k, is needed to convert this value into a
The gradient chosen for sensitivity measurements and the
force F, using the well-known Hooke's law: baseline offset for the deflection are both marked on this plot.
F kx When the cantilever is far from the surface, the interaction

forces are virtually zero (the flat part of the curve on the
A force curve between a plain cantilever tip and a bare right hand side in Figure 4). This offset (which may be
hard substrate is used to determine the sensitivity of the due to the initial settings of the equipment, or to thermal
experimental setup. This is a measurement of the drift) should be subtracted from all the deflection data in
deflection of the tip in nanometers for a given movement of order to calculate the true interaction force. The baseline
the detection laser on the photodetector. The repulsive value is also marked in Figure 4. Note that the very flat
contact region, where the deflection rises steeply upwards, baseline is due to the suppression of optical interference
is linear for a hard surface and tip. Therefore the software effects by the state-of-the-art detection system employed
can easily determine the factor for converting Volts into in the NanoWizard AFM.
nanometers. This measurement can then be used for
calibrating the applied forces when the samples of interest For older systems or homebuilt equipment it is important to
are investigated. The sensitivity can then also be used to take account of the piezo hysteresis in these
set the oscillation amplitude in intermittent contact mode as measurements, in order to get an accurate value for the
actual nanometers of oscillation. sensitivity. When a piezo moves over a relatively large
range, nonlinearity and hysteresis are problems that can
Since the hard repulsive interaction regime is used for the lead to false data for the cantilever position. These
sensitivity measurement, it should usually be performed at problems can be overcome by using a separate system to
the end of an experiment, to avoid damaging the tip. simultaneously measure the z-piezo position during the
Figure 4 shows part of a force curve from a plain contact movement, such as the precision sensors used in the JPK
mode cantilever on mica under aqueous buffer solution NanoWizard AFM. This measured z value is independent
(PBS). The data here is the "raw" information, as cantilever of the piezo nonlinearity and hysteresis.
deflection in displayed in Volts. The gradient of the
page 4/8
The benefits of piezo linearization can be seen in Figure 5, deflection of the cantilever can be converted into actual
where the same force measurement is shown with force values. There are various different ways of calibrating
linearized (A) and unlinearized (B) height scales. In the spring constants of cantilevers, depending on the
upper image (A), where the sensor height values are used, equipment that is available, and only the three most
the approach and retract curves exactly overlay, as they common methods are described here. Each method is
should for a hard clean sample surface. The natural piezo subject to some limitations, and if the force measurement
hysteresis can be seen in the curves in the lower panel (B). is particularly important, the spring constant should be
calibrated using more than one method to verify the
results.
A Linearized piezo (height is measured)
approach and retract are identical
Calculation from the cantilever geometry
When cantilevers are purchased, they are usually supplied
Approach with a data sheet, which includes the spring constant. The
values are generally given as a range, and may have been
Retract calculated from the average cantilever geometry, rather
than having been experimentally measured for each
cantilever. It is possible to calculate the spring constant, if
the exact shape of the cantilever, and the Young's modulus
of the material is known.
B Unlinearized piezo (height from piezo A problem with this method is that the spring constant
voltage) approach and retract show depends on the thickness of the cantilever cubed. This
hysteresis
value is also the smallest dimension of the cantilever.
Therefore the spring constant is extremely sensitive to
Approach
slight differences in cantilever thickness between batches
of cantilevers. The quoted spring constant values from
Retract
manufacturers are useful for choosing a cantilever for a
particular application, but are usually not reliable for
quantitative force measurements.
Fig. 5 Comparison of results obtained with piezo linearization (A)

and without piezo linearization (B). Plots of approach (red) and
Measurement using a reference cantilever
retract (blue) curves using a contact mode cantilever and clean When a reference cantilever with a known spring constant
mica in water. is available, other cantilever spring constants can be
calculated by comparing the deflection of the two
The curves in Figure 5 show the converted data from the cantilevers when they are pushed together [2]. The
experiment in Figure 4. The baseline has been shifted to unknown cantilever can be mounted as normal in the AFM,
take account of the deflection offset far from the surface, and the reference cantilever is used as the "sample". The
and the sensitivity value has been used to convert the cantilevers are approached to each other so that they just
deflection into nanometers. The deflection values in Figure overlap at the end, and a force curve is performed.
5A are now ready to be converted to units of force.
The slope of the repulsive contact part of a force-distance
Cantilever spring constant calibration curve on a hard surface and on the reference cantilever
For quantitative force measurement, the spring constant of must be compared. The deflection of the unknown
the cantilever must be calibrated, so that the nanometers cantilever compared with the reference cantilever gives a
page 5/8
conversion for the spring constant. This calculation is most measurement and automatic spring constant calculation is
accurate if the two cantilevers have similar values. The offered as standard in the JPK SPM software, so this can
method is relatively straightforward if the AFM is equipped be performed without any extra equipment.
with a good optical imaging system, so that the cantilevers
can be well aligned, but properly calibrated reference Force conversion and resolution
cantilevers are expensive. When the deflection of the cantilever is known in units of
length, as in Figure 5A, then it is straightforward to convert
Measurement using the thermal noise to force units. The deflection (in meters) is multiplied by the
method spring constant of the cantilever (in N/m) to give a force in
Fluctuations in the environment (in air or fluid) constantly Newtons. For molecular interactions, forces are usually
provide small force impulses, as can be seen for example seen in the range of picoNewtons (1 pN = 10-12 N) to
in the diffusion of small particles (Brownian motion). Soft nanoNewtons (1nN = 10-9 N).
cantilevers are susceptible to thermal fluctuations, and the
AFM can be used to measure and analyse the movements. So far, all the conversion has been in the y-axis, or
The thermal noise spectrum is a plot of the cantilever deflection/force values. There is also one conversion
fluctuations as a function of frequency; on average the needed for the height, or x-axis values, however. The
greatest amplitude will be seen around the cantilever change in piezo height has been used for the distance
resonance frequency. An example is shown in Figure 6. between the tip and the sample, but in fact this must be
Longer integration times of the cantilever motion give corrected for the deflection of the cantilever. If the
better signal to noise in the spectrum. cantilever deflects towards the sample, for example, then
the separation between the tip and the surface will be less
than expected from the piezo position alone. Since the
cantilever deflection and position are available in the same
units, this can be easily corrected by subtracting the
cantilever deflection from the piezo height.
Approach
Retract
Fig. 6 Thermal noise measurement using the JPK software of a

contact mode cantilever in air, showing the measured cantilever
resonance peak (blue) and the Lorentz fit (red). The fit in this
case gave a spring constant of 0.147N/m. Fig 7. Force-separation (corrected height) plot for the data shown
in Figures 4-5.
The amplitude of the fluctuations for a given temperature
depends only on the spring constant of the cantilever [3]. This conversion leads to the gradient of the steep upwards
The thermal resonance curve can therefore be fitted to a part of the curve becoming vertical. Once the tip is in
Lorentz function, which allows calculation of the spring contact with the surface, the tip-sample separation remains
constant. This method has the advantage that it can be at zero while the force is increased by the cantilever
done in-situ with software analysis. Thermal noise pushing towards the surface. An example of the final
page 6/8
converted curve is shown in Figure 7. Here the x-axis is normal force spectroscopy experiments. Here, for
plotted as the actual tip-sample separation, rather than the example, the maximum applied force is much lower than in
piezo movement and the deflection values are now the measurements for sensitivity calibration in Figures 4-7.
displayed in units of force. The data here would now be To show the force resolution, a part of the baseline in
ready for analysis. Figure 8 is shown with a magnified height scale in the inset
curve.
One important consideration for force measurements is the
smallest detectable force. This is affected by many
different factors, and the properties of the cantilever itself
play an important role in determining the force resolution.
In the AFM instrument, the deflection of the cantilever is
measured by the optical detection system and there will be
a limit on the smallest detectable deflection. For softer
cantilevers, this limit on the detectable deflection
corresponds to a lower force value. Therefore it might
Approach
seem that the force resolution should always be improved
by using a softer cantilever. Retract
When the forces and distances involved correspond to the Fig 8. Force-separation plot for a 10pN/nm cantilever in buffer. A
thermal energy, however, the thermal fluctuations of the part of the baseline is displayed in a magnified view in the inset
curve, to show the resolution limit of the force data due to thermal
cantilever become significant. The scale for thermal
fluctuations.
energy at room temperature can be calculated by using the
Boltzmann constant, so for example at 25C, kBT = 4.1 x
Advanced force measurement applications
10-20 J. The units of energy correspond to units of force Over a soft, compressible sample in liquid, such as a gel or
times units of distance, so 1 Joule = 1m x 1N. It can be a living cell, the overall shape of the force curves is often
helpful to express the thermal energy in units that are more quite different from the hard-surface examples shown here.
appropriate to single molecules, for example as kBT = 41 The curves are more likely to show a gradual increase in
pN nm. The spring constants for the cantilevers can also force, without the sharp onset of the interactions seen in
be expressed in more helpful size ranges; for example, a air. The gradient of the repulsive contact region may not be
soft cantilever for force spectroscopy with a spring constant, as the sample stiffness (change in indentation
constant value of 0.01 N/m is equivalent to 10 pN/nm. A depth for a given change in force) may change as the
force of 10pN would therefore cause a deflection of 1 nm structure is compressed. For thin samples on a hard
for this cantilever. surface, the linear repulsive contact regime may be seen at
large deflections, as the tip may indent the sample enough
Softer cantilevers have larger thermal fluctuations, and the to feel the supporting surface below.
soft cantilevers used for many single-molecule force
spectroscopy experiments have significant thermal The tip-sample contact area will change as the tip indents
fluctuations. In this case, the force resolution is usually a soft surface, so sometimes a larger object with a well-
limited by the thermal noise of the cantilever. The effective known shape is attached to the tip. This may be a micron-
limit on the force resolution for a particular experiment can sized sphere or bead, such as the fluorescent
be seen from the noise on the deflection baseline. microspheres used as calibration standards. This gives a
more defined and reproducible contact area for a given
An example curve for a soft cantilever (10 pN/nm spring indentation depth. This can be useful for indentation
constant) is shown in Figure 8, for parameters closer to the
page 7/8
experiments, and also for adhesion measurements if single external electrical potential, information about magnetic
molecule experiments are not the aim. and electric properties of the sample can be obtained.
When the tip is retracted from the surface, there is often a Varying the speed of the tip movement can give further
hysteresis observable, if the sample is not perfectly elastic, information about the sample responses. The approach
since on the retract movement the surface has already and retract rates can be varied independently, and waiting
been compressed. If the piezo hysteresis has been times added at the surface or at the retracted position.
eliminated (as, for example, in Figure 5A), then this sample This flexibility allows exploration of the time-dependent
hysteresis can be measured accurately for different response of the sample, for example adsorption times or
approach and retract rates. on-rates for binding, relaxation times for mechanical
properties, or re-folding rates for proteins.
Different types of adhesion events can be seen, depending
on what links the tip and the sample. If long molecules A detailed review of force spectroscopy applications is
connect the tip and sample, then the stretching of these outside the scope of this technical note. The steps outlined
tethers produces characteristic curved sections on the in this report to convert the cantilever deflection into
retract deflection curves. When the molecule is pulled free quantitative force measurements are the starting point for
from either the tip or sample, or unfolds or even breaks any more complex analysis. The NanoWizard hardware
somewhere between, the deflection jumps sharply as the and software provides the perfect combination to study
tip is freed. Multiple events may be seen as connections of forces on the nano scale.
different lengths are stretched and broken as the tip
retracts.
Literature
Other physical properties can also be measured, as well as [1] Janshoff A., Neitzert M., Obersrfer Y., and Fuchs H. "Force
Spectroscopy of molecular systems single molecule
the actual interaction force [4]. Electrical signals, such as
spectroscopy of polymers and biomolecules". Angew. Chem.
the conductivity through the tip and sample, or external Int. Ed. 39:3212-3237 (2000).
properties such as fluorescence can be monitored during [2] Hinterdorfer et al. Detection and localization of individual
the tip movement. In dynamic force spectroscopy antibody-antigen recognition events by atomic force
microscopy. PNAS 93 3477-3481 (1996).
experiments the cantilever is oscillated during the [3] Hutter J.L. and Bechhoefer J. " Calibration of atomic-force
movement, and the oscillation amplitude and phase signals microscope tips". Rev. Sci. Instrum. 64:1868-1873 (1993).
can be measured. This can give information about the [4] Jena B.P. and Hrber J.K.H. "Atomic force microscopy in cell
biology" Methods in Cell Biology Vol. 68 (2002).
viscoelastic properties (elastic and dissipative response) of
the sample. If this is combined with a magnetized tip or an
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A practical guide to AFM force spectroscopy and data analysis

Tip approaching Electrostatic forces

Tip close to surface Van der Waals

Tip further away Stretched molecules between tip and surface:

Other stretched attachments e.g. membrane tethers formed on cells

Understanding simple force curves

Calibration of the cantilever deflection Approach

F kx When the cantilever is far from the surface, the interaction

Fig. 5 Comparison of results obtained with piezo linearization (A)

Fig. 6 Thermal noise measurement using the JPK software of a

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